**3.1 The effect of substrate type and temperature on the isomerization reaction of glucose isomerase**

The equilibrium reaction from fructose to glucose or vice versa took place regardless of substrate type and temperature. According to the results of the experiments, the isomerization reactions were equilibrated at a faster rate when starting with glucose compared with fructose, especially at 30°C. Another important point resulting from experiments, the reactions took place at faster rates when conducted at 60°C compared with 30°C. Also, an equilibrium point was reached after 5 hours at 60°C while after 9 hours at 30°C (data not shown). As a result of these

experiments, it can be understood that the enzyme is suitable for both substrate types at different temperatures.

#### **3.2 The effect of ethanol on the isomerization reaction of glucose isomerase**

According to the results, glucose formation and fructose depletion were shown at 60 and 30°C. Also, it was shown that glucose concentration was slightly higher than fructose concentration after 25 hours at 30°C. However, fructose concentration was slightly higher than glucose concentration after 25 hours at 60°C. As it was mentioned before, the reaction took place more slowly at 30°C compared with 60°C by looking at the data (data not shown). As a consequence of this experiment, it was interpreted that 13% v/v ethanol did not inhibit the activity of the enzyme regardless of different temperature values.

Since ethanol did not inhibit the isomerization reaction in synthetic environment, an experiment was conducted by vaporizing ethanol in red wine to show the effect of ethanol on red wine. The ethanol concentration was reduced approximately from 13 to 0.3% v/v by keeping wine at air temperature on the magnetic stirrer. At the end of 42 hours, glucose concentrations of four samples did not change (data not shown). This means that the enzyme did not isomerize fructose even if there was no ethanol in the wine.

#### **3.3 The effect of low pH values on isomerization reaction in synthetic medium**

As seen in **Figure 1**, it was noticed that fructose was converted to glucose regardless of the pH values. Also, it was clearly noticed that the reaction at pH 4 was the fastest one when compared to others, whereas the slowest reaction was at pH 3.3 by looking at glucose formation rate. Therefore, it was thought that the reason for the inhibition of enzyme in the wine medium was not because of the low pH values. The rate of reaction decreased proportionally with decreasing pH, the inhibition effect in the wine medium was not due to acidic medium, and the effect of ethanol with low pH values had to be tested.

Under the same conditions in **Figure 1**, the glucose was formed under the same pH values with 13% v/v ethanol in medium as seen in **Figure 1**, too. The ethanol

**Figure 1.** *Change of glucose concentrations at different pH values without and with ethanol.*

*Determining Glucose Isomerase Activity in Different Wine Environments to Prevent Sluggish… DOI: http://dx.doi.org/10.5772/intechopen.100499*

effect experiments were conducted with the same amount of ethanol as experiments above, 13% v/v. The rate of reaction was obviously slower at pH 3.3. When without and with ethanol conditions were compared, almost the same amounts of glucose were formed for pH values of 3.6 and 4, whereas the amount of glucose at pH 3.3 was clearly lower in ethanol medium. Also, the glucose amounts formed at pH 3.6 and 4 were equalized at about the 45th hour with ethanol in the environment. The formed glucose amounts from 1% w/v fructose were nearly 0.2 and 0.1% w/v in medium without and with ethanol, respectively, at pH 3.3. As a consequence, glucose formation was detected in acidic media even in the presence of ethanol. The rate of reaction at pH 3.3 was clearly slower than others regardless of the presence of ethanol.

## **3.4 The effect of glycerol in synthetic media on the isomerization reaction**

According to **Figure 2**, the isomerization reaction took place in all synthetic media with different glycerol concentrations. The rates of reactions were close to each other for all glycerol concentrations until the glycerol concentration was approached to 1% v/v. That is, the slowest reaction rate was obtained at the concentration of 1% of glycerol. It was easily concluded that the existence of glycerol in synthetic media containing ethanol did not inhibit the isomerization reaction.

#### **3.5 The effect of sulfite content on isomerization reaction**

Although it is known that glucose isomerase is used with sulfite [13], the experiments were also conducted in synthetic media with sulfite. After 14.5 hours of incubation, almost equal amounts of glucose were formed in all concentrations: 0.1, 0.04, and 0.01%, of sulfite (data not shown). Therefore, even higher sulfite contents compared to those present in wine did not inhibit the activity of glucose isomerase.

#### **3.6 The effect of tannins in red wine on the isomerization reaction**

It was thought that tannins might have an inhibitory effect on the isomerization reaction. Therefore, the comparison of glucose isomerase activity in red and white wine media was made. Glucose concentrations of all types of wines, w1, w2, and w3 remained stable during 70 hours of incubation. Normally, enzyme activity is seen in the first 2 or 3 hours of the experiments; in this case, no activity was observed even after 70 hours (data not shown). As a consequence, it was concluded that tannins had no inhibitory effect on the isomerization reaction since white wine also showed a similar behavior.

**Figure 2.** *Change of glucose concentrations in synthetic media with different glycerol contents during 47 hours.*
